Compositions and methods for laser capture microdissection

ABSTRACT

Compositions and methods for the simultaneous capture and release using micropattern surfaces for tissue and cell microdissection. In one example, a patterned thermoplastic film has a first surface and a plurality of projections attached to and extending outwardly from the first surface. The projections form a pattern on the thermoplastic film.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims priority to and the benefit of the filing dateof U.S. Provisional Patent Application No. 62/271,027, filed Dec. 22,2015, the content of which is hereby incorporated by reference herein inits entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number1R33CA173359-03 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Laser Capture Microdissection (“LCM”) is an established technology usedto pluck out tumor cells, or other types of cells, from a heterogeneouspiece of tissue under direct microscopic visualization. The procuredcells are used in commercial diagnostic assays, clinical trials, andresearch studies by pharma and academia. LCM is used by thousands ofscientists worldwide. The most popular, and most useful, form of LCMemploys a laser beam or a source of radiation to heat a flat plasticfilm that is held against the slice of tissue mounted on a glass slide(FIG. 1A). The plastic film is uniformly impregnated with a dye thatabsorbs laser energy. The region of the plastic film positioned over thetissue region or cell of interest is selectively heated by the radiationcausing this region to melt and embed itself into the tissue segmentimmediately underneath (FIG. 1B). When the film is lifted off the tissuethe portions of the tissue adherent to the undersurface of the film areripped free of the tissue section (e.g., see Espina V., et al. (2006)Nature Prot. 1(2):586-603).

Despite the power and utility of LCM, the method is currently limited bya number of shortcomings. First, currently available LCM methods sufferfrom the variability in the capture efficiency of the desired region ofthe tissue. The variability is caused by a reduced adhesive force on thetop of the tissue section between the tissue and the film and anincreased adhesive force between the tissue section and the glass slidesubstratum. Second, the resolution of LCM systems is limited because thefilm melts against the tissue in a variable manner depending on thefocus of the laser beam, the surface contours, and the wavelength of thelight. An area spanning many tissue cell diameters is often captured,thus limiting the resolution and preventing the precise capture ofsingle cells or components of cells.

Despite advances in methods and composition pertaining LCM, there remainsignificant drawbacks to the methods which limit the use and applicationof LCM. These needs and other needs are satisfied by the presentdisclosure.

SUMMARY

In accordance with the purpose(s) of the disclosure, as embodied andbroadly described herein, the disclosure, in one aspect, relates tocompositions and methods for laser capture microdissection. In variousaspects, the present disclosure pertains to compositions and methods forthe simultaneous capture and release using micropattern surfaces fortissue and cell microdissection.

Disclosed herein are patterned (e.g., micropatterned) thermoplasticfilms comprising projections that are attached to, continuous with, orintegrally formed with a surface of the micropatterned thermoplasticfilm. In various aspects, a patterned thermoplastic film can have afirst surface and a plurality of projections secured to and extendingoutwardly from the first surface, with the projections forming a patternon the thermoplastic film.

Also disclosed are methods for laser capture microdissection, comprisingthe steps of: (a) placing a disclosed micropatterned thermoplastic filmin contact with a tissue sample, and (b) irradiating the micropatternedthermoplastic film in contact with the tissue sample withelectromagnetic radiation in the UV or IR spectrum.

Also disclosed are kits including a disclosed micropatternedthermoplastic film and instructions for using the film in a lasercapture microdissection. In an aspect, it is contemplated that the kitscan further comprise ingredients for producing the micropatternedthermoplastic film. In another aspect, it is contemplated that the kitscan further comprise dyes and/or applicators for producing or using thedisclosed micropatterned thermoplastic films.

While aspects of the present disclosure can be described and claimed ina particular statutory class, such as the system statutory class, thisis for convenience only and one of skill in the art will understand thateach aspect of the present disclosure can be described and claimed inany statutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the disclosure.

FIG. 1A depicts a typical LCM system, which includes a thermoplasticfilm placed in contact with an upper surface of tissue, which has anundersurface that is mounted on a glass slide. FIG. 1B schematicallydepicts activation of the thermoplastic film by laser enerty, causingthe film to locally melt and become embedded at the tissue surface.

FIGS. 2A-2C show exemplary components of the disclosed apparatus, whichincludes a patterned thermoplastic film. FIG. 2A depicts a patternedthermoplastic film prior to contact with tissue as disclosed herein.FIG. 2B depicts the patterned thermoplastic film in contact with thetissue as UV or IR laser irradiation is applied. FIG. 2C shows anexpanded close-up view of the area within the oval of FIG. 2B.

FIGS. 3A and 3B show configurable aspects of projections of patternedthermoplastic film as disclosed herein. FIG. 3A schematically depicts anincrease in adhesive contact with tissue at the tip of a projection asdisclosed herein. FIG. 3B schematically depicts the activation of“microneedle” projections as disclosed herein to form an expanded tipanchor.

FIGS. 4A-4C show configurable aspects of hydrogel nanoparticlespositioned on projections of a patterned thermoplastic film as disclosedherein. FIG. 4A schematically depicts non-adhesive nanoparticles, whichare shed from an activated projection or tip of the micropatternedsurface as the projection expands and contacts the tissue upper surfacebut not the the lower tissue surface. FIG. 4B schematically depicts thebreaking of the shell of an adhesive nanoparticle to make thenanoparticle non-adhesive, thereby dissociating crosslinks in the shellin response to interaction with an IR or UV laser beam. FIG. 4Cschematically depicts the breaking of the hydrophobic non-adhesive shellof a nanoparticle in response to interaction with an IR or UV laser beamto expose an inner adhesive core.

FIGS. 5A-5D show representative components of exemplary fabricatedmicropattern surfaces. FIGS. 5A and 5B show representative fabricatedmicropattern surfaces that were produced by an inverted photolithographymold applied to the thermopolymer surface to form the micropatternsurface. FIG. 5C is an image of a standard cap component where thethermopolymer surface was mounted. FIG. 5D shows that that tips ofprojections (e.g., micropillars) formed by these methods can be wettedby water while the base remains repulsive and non-wetted by the water.FIGS. 5E and 5F are representative microscopic images of captured cellsusing the micropattern surfaces of FIGS. 5A and 5B, respectively.

Additional advantages of the disclosure will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the disclosure. Theadvantages of the disclosure will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the disclosure, as claimed.

DESCRIPTION

The present disclosure can be understood more readily by reference tothe following detailed description of the disclosure and the Examplesincluded therein.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a projection” caninclude two or more such projections unless the context indicatesotherwise.

All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

The purpose of the laser capture microdissection (“LCM”) technique is toprovide a simple method for the procurement of selected human cells froma heterogeneous population contained on a typical histopathology biopsyslide. A typical tissue biopsy sample consists of a 5 to 10 micron sliceof tissue that is placed on a glass microscope slide using techniqueswell known in the field of pathology. This tissue slice is a crosssection of the body organ that is being studied. The tissue consists ofa variety of different types of cells. Often a pathologist desires toremove only a small portion of the tissue for further analysis.

FIGS. 1A and 1B show typical LCM components. As shown in FIGS. 1A and1B, the components can include a thermoplastic film 120 that is placedin contact with an upper surface 130 of the tissue 125, while theundersurface 140 of the tissue is mounted on a glass slide 50.Activation of the film by laser energy 110 causes the film to locallymelt an area 121 beneath the thermoplastic film 120 that is in the beamof the laser energy 110. The melted area of the film 121 embeds at thetissue surface. The film 120 is then removed and thereby tears away thetissue portion immediately below the locally melted film area 121. Thesuccess of this operation is dependent on the balance of forces abovethe selected region of interest (in region “U”) and below the selectedregion of interest (in region “B”).

In various aspects of the present disclosure, LCM can be employed usinga patterned (e.g., micropatterned) thermoplastic transfer filmcomprising projections, such as micropillars, micro projections,hydrogel microspheres, and/or microneedles as further disclosed herein,that are attached to, continuous with, or integrally formed with athermoplastic film, that is placed on top of the tissue sample. Thisfilm and/or the projections can be manufactured containing organic dyesthat are chosen to selectively absorb in the ultraviolet or infraredregion of the spectrum overlapping the emission region of common laserdiodes, e.g., AlGaAs laser diodes. In an aspect, when the film isexposed to the focused laser beam the exposed region is heated by thelaser and melts, adhering to the tissue in the region that was exposed.The film is then lifted from the tissue and the selected portion of thetissue is removed with the film. As further disclosed herein, it iscontemplated that the disclosed compositions and methods can increasethe adhesive strength and improve the resolution of the film in contactwith the tissue surface while at the same time selectively reducing theadhesive forces on the bottom of the tissue, where it can be tightlydried down on the glass slide.

In an aspect, and with reference to FIGS. 2A-4A, a micropatternedthermoplastic film 70 comprises projections 72, such as micropillars,micro projections, hydrogel microspheres, and/or microneedles that areattached to, continuous with, or integrally formed with a thermoplasticfilm layer 71 that defines a first (e.g., bottom) surface 76. In afurther aspect, the projections 72 (e.g., micropillars, microprojections, hydrogel microspheres, and/or microneedles as disclosedherein) form an array on a surface (e.g., the first surface 76) of themicropatterned thermoplastic film. In some aspects, the projections 72(e.g., micropillars, micro projections, hydrogel microspheres, and/ormicroneedles) are formed on a single surface of a planar orsubstantially planar thermoplastic film layer 71, and an opposingsurface of the substantially planar thermoplastic film layer can besubstantially smooth or lacking projections (e.g., micropillars, microprojections, hydrogel microspheres, and/or microneedles). Optionally, asshown in FIGS. 2A-2C, the projections 72 can extend outwardly from thefirst surface 76 of the patterned thermoplastic film layer 71 relativeto an axis 78 that is perpendicular or substantially perpendicular tothe first surface. As used herein, the term “micropatterned” refers tothermoplastic films that are patterned with projections as furtherdisclosed herein.

In an aspect, the micropatterned thermoplastic film 70 can comprise anarray of projections 72 that cooperate to define a pattern on a surfaceof the film. In this aspect, it is contemplated that the projections 72of the array can comprise micropillars, micro projections, hydrogelmicrospheres, microneedles, or combinations thereof. As used herein, theterm “micropillar” refers to a projection that, prior to activation asdisclosed herein, has a consistent or substantially consistent outerdiameter along its length. As used herein, the term “microsphere” refersto a projection that, prior to activation as disclosed herein, has agenerally rounded appearance, including spherical, substantiallyspherical, ovoid, and substantially ovoid shapes. As used herein, theterm “microneedle” refers to a projection that, prior to activation asdisclosed herein, has a diameter that decreases (optionally,consistently decreases) moving away from the first surface 76 of thepatterned thermoplastic film. It is contemplated that the “micropillars”and “microneedles” can have any desired cross-sectional shape,including, for example and without limitation, circular, square,rectangular, triangular, oval, elliptical, trapezoidal, pentagonal,hexagonal, heptagonal, or octagonal shapes. Optionally, as shown in FIG.3B, a microneedle can have a pointed distal tip. In an aspect, at leastone of the projections of the array can have a consistent diameter.Optionally, in this aspect, at least two of the projections can have aconsistent diameter. In a further aspect, each projection of theplurality of projections can have a consistent diameter. In anotheraspect, at least one of the projections can have a variable diameterthat increases or decreases moving away from the first surface 76 of thepatterned thermoplastic film. Optionally, in this aspect, at least twoof the projections can have a variable diameter. In a further aspect,each projection of the plurality of projections can have a variablediameter. In one exemplary aspect, it is contemplated that eachprojection of the plurality of projections can be a micropillar. Inanother exemplary aspect, it is contemplated that each projection of theplurality of projections can be a microsphere. In another exemplaryaspect, it is contemplated that each projection of the plurality ofprojections can be a microneedle. In still another exemplary aspect, itis contemplated that the plurality of projections can comprise at leastone micropillar and at least one microneedle. In still another exemplaryaspect, it is contemplated that the plurality of projections cancomprise at least one micropillar and at least one microsphere. In stillanother exemplary aspect, it is contemplated that the plurality ofprojections can comprise at least one microsphere and at least onemicroneedle. In still another exemplary aspect, it is contemplated thatthe plurality of projections can comprise at least one micropillar, atleast one microneedle, and at least one microsphere.

In an aspect, the projections 72 of the micropatterned thermoplasticfilm 70 can have a width or outer diameter ranging from about 1 nm toabout 10 mm and a length ranging from about 1 nm to about 10 mm. As onewill appreciate, the length of each projection 72 can correspond to theheight of the projection relative to the first surface 76 of themicropatterned thermoplastic film 70. In exemplary aspects, the width orouter diameter and the length of each projection can both range fromabout 100 nm to about 1 mm. Thus, in these aspects, it is contemplatedthat the width or outer diameter can be about 100 nm, about 200 nm,about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm,about 800 nm, about 900 nm, about 1 μm, about 10 μm, about 20 μm, about30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm,about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm,about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm,about 190 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm,about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm,or can fall within a range defined between any two of these listedvalues. Optionally, in exemplary aspects, it is contemplated that thelength of each projection 72 can be greater than the width or outerdiameter of the projection. In one exemplary aspect, the projections 72can each have a diameter of about 100 μm, and the projections can befabricated such that each of the projections has a consistent (equal orsubstantially equal) height ranging from about 10 μm to about 100 μm.

During fabrication of the pattern, it is contemplated that theprojections 72 can be fabricated to have any desired spacing relative toother projections (e.g., adjacent projections) within the array.Optionally, in some aspects, the projections 72 can have an equal orsubstantially equal spacing within the array (measured between centerpoints of the respective projections). For example, it is contemplatedthat the projections 72 can be organized in columns, rows, orcombinations thereof, in which each projection is spaced from adjacentprojections by a consistent distance, such as, for example and withoutlimitation, a distance ranging from about 10 μm to 1 mm and, morepreferably, from about 10 μm to about 200 μm, or about 100 μm. However,in other aspects, it is contemplated that the projections can befabricated to have a variable spacing, with at least one projectionbeing closer to some adjacent projections than others. In these aspects,it is contemplated that portions of the array can have a higherconcentration of projections than other projections. It is furthercontemplated that the array of projections can include at least oneprojection having a width or outer diameter that is different than thewidth or outer diameter of at least one other projection. Optionally, itis contemplated that the projections can include projections havingthree or more different widths or outer diameters. It is still furthercontemplated that the array of projections can include at least oneprojection having a length that is different than the length of at leastone other projection. Optionally, it is contemplated that theprojections can include projections having three or more differentlengths. As used herein, in the context of spacing between projections,“adjacent” does not indicate physical contact; instead, the term“adjacent” refers to the projection in closest proximity to a givenprojection relative to a particular reference axis. For example, ifprojections are arranged in a row, then a selected projection may beadjacent to the two projections within the row on either side of theselected projection. If the selected projection is also arranged withina column of projections, then the selected projection may also beadjacent to the two projections within the column on either side of theselected projection.

In an aspect of the present disclosure, the projections are formed on afilm using a photolithography mold that is applied to the thermopolymersurface mounted on a cap. In a further aspect, the micropatternedthermoplastic transfer film can be a film such as a 100 micron thickethyl vinyl acetate (EVA) film available from Electroseal Corporation ofPompton Lakes, N.J. (type E540), on which projections have been formed,e.g., using a photolithography mold. The film is chosen to have asuitable melting point, e.g., from about 70° C. to about 120° C. In anaspect, the melting point of the film is about 90° C.

The micropattern thermoplastic films of the present disclosure can beany suitable thermoplastic. For example, the micropattern thermoplasticfilm can be fabricated from one or more of: EVAs; polyurethanes (PU);polyvinyl acetates; ethylene-methyl acrylate (EMAC); polycarbonate (PC);ethylene-vinyl alcohol copolymers (EVOH); polypropylene (PP); andexpandable or general purpose polystyrene (PS). ELVAX 410, 200 and 205are suitable resins of EVA that are commercially available from DuPontwherein the operative variant is the amount of vinyl.

In an aspect, micropatterned thermoplastic film, such as an EVA film,used in LCM techniques can comprise an absorptive substance. Theabsorptive substance can include an absorptive dye. This dye can beeither a broad band absorptive dye or a frequency specific absorptivedye. For example, the absorptive dyes can include one or more of:tin(IV) 2,3-naphthalocyanine dichloride; silicon(IV)2,3-naphthalocyanine dihydroxide; silicon (IV) 2,3-naphthalocyaninedioctyloxide; and vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine. The absorptive dye canbe an infrared napthalocyanine dye, available from Aldrich ChemicalCompany (dye number 43296-2 or 39317-7). Also, the absorptive substancecan include a plurality of fullerines (i.e., Bucky Balls, e.g., C60). Anabsorptive substance can have a strong absorption in the 800 nm region,a wavelength region that overlaps with laser emitters used toselectively melt the film. The absorptive substance is mixed with themelted bulk plastic at an elevated temperature. The thermoplasticcomprising the absorptive substance is then manufactured into a filmusing standard film manufacturing techniques. The dye concentration inthe plastic can be about 0.001 M.

The micropatterned thermoplastic film of the present disclosure canfurther comprise a scattering media. Since the micropatternedthermoplastic film is very close to the sample, the scattering mediareduces shadows, and can thereby improving the process of imaging. Thescattering media can include a diffusing material. For example, themicropatterned thermoplastic film can be loaded with a small particulatematerial that scatters the illumination light so as to minimize shadowsand improve imaging without detrimentally effecting the LCM beam.Alternatively, the micropatterned thermoplastic film can include adichromatic gelatin (DCG) to perform the same functions. The DCG can beexposed and developed to provide specific diffuser properties within thetransfer film such as shaping.

The micropatterned thermoplastic films of the present disclosure areused with appropriate laser sources. For example, suitable laser pulsewidths are from 0 to approximately 1 second, preferably from 0 toapproximately 100 milliseconds, more preferably approximately 50milliseconds. In a preferred embodiment the wavelength of the laser is810 nm. In a preferred embodiment the spot size of the laser at the EVAmaterial located on microcentrifuge tube cap 120 is variable from 0.1 to100 microns, preferably from 1 to 60 microns, more preferably from 5 to30 microns. These ranges are relatively preferred when designing theoptical subsystem. From the standpoint of the clinical operator, thewidest spot size range is the most versatile. A lower end point in thespot size range on the order of 5 microns is useful for transferringsingle cells.

Suitable lasers can be selected from a wide power range. For example, a100 watt laser can be used. On the other hand, a 50 mW laser can beused. The laser can be connected to the rest of the optical subsystemwith a fiber optical coupling. Smaller spot sizes are obtainable usingdiffraction limited laser diodes and/or single mode fiber optics. Singlemode fiber allows a diffraction limited beam.

Changing the beam diameter permits the size of the portion of the samplethat is acquired to be adjusted. Given a tightly focused initialcondition, the beam size can be increased by defocusing. Given adefocused initial condition, the beam size can be decreased by focusing.The change in focus can be in fixed amounts. The change in focus can beobtained by means of indents on a movable lens mounting and/or by meansof optical glass steps. In any event, increasing/decreasing the opticalpath length is the effect that is needed to alter the focus of the beam,thereby altering the spot size. For example, inserting a stepped glassprism 380 into the beam so the beam strikes one step tread will changethe optical path length and alter the spot size.

As shown in FIGS. 2A-2B, the disclosed apparatus can replace twocomponents of the conventional LCM capture technology with newmaterials. As further disclosed herein, the top film of conventional LCMtechnology, which is flat and smooth, can be replaced by amicropatterned thermoplastic film comprising projections having surfacesthat can be activated by selective radiant energy, such as from a laserbeam, to become adhesive to the irregular tissue surface 30 below.Additionally, as further disclosed herein, the glass slide 50 that thetissue is mounted on can also be modified by coating it withmicroparticles or nanoparticles that can be altered in their tissueadhesive properties by UV or IR laser irradiation (FIG. 2A) When thelaser irradiation takes place over the desired region of the tissue 25,the adhesive capture is activated above the tissue and dissolved belowthe tissue (FIGS. 2B and 2C). This addresses both sources of variabilityto achieve a higher efficiency, yield, and resolution, with no changesto the overall operation of the other components of the LCMinstrumentation.

FIGS. 2A-2C show exemplary components of the LCM of the presentdisclosure. FIG. 2C shows an expanded view of the area within the ovalof FIG. 2B. As shown in FIGS. 2A-2C, the smooth film 120 depicted inFIGS. 1A and 1B, which is typically used, is replaced by amicropatterned thermoplastic film 70 comprising projections 72, such asmicropillars, micro projections, hydrogel microspheres, and/ormicroneedles, that are attached to, continuous with, or integrallyformed with a thermoplastic film surface 71. The projections can conformto the upper tissue surface 30 and increase the adhesive forces to theirregular tissue surface below when activated by UV or IR laserirradiation 10. The increased adhesive forces are due, at least in part,to an expanded projection tip 73 that occurs upon activation by UV or IRlaser irradiation 10. The laser irradiation is directed over the desiredregion of tissue 25, and the laser irradiation activates the adhesivecapture above the tissue. The lower surface 40 of the tissue 25, insteadof directly resting on the glass slide 50, rests on a microparticle ornanoparticle coating 80 that can be altered in its adhesive propertiesby the laser energy alone or in combination with a chemical treatment 81of the tissue 25. The laser energy simultaneously activates the adhesiveforces on the top face while breaking or dissolving the adhesive forcebelow 81. This achieves high efficiency and precision of capture withlower non specific capture.

FIGS. 3A and 3B show configurable aspects of the micropatterned surfaceprojections 72. In both FIGS. 3A and 3B, the micropatternedthermoplastic film 70 comprises projections 72, such as micropillars,micro projections, hydrogel microspheres, and/or microneedles, that areattached to or continuous with a thermoplastic film layer 71 asdisclosed herein. The projections 72 can be impregnated with a UV or IRabsorbing dye in a gradient concentrated at the distal tip of theprojections or pillars. In one aspect, as shown in FIG. 3A, activationby the laser can expand and increase the adhesive contact with thesurface only at the tip 74. In an alternative aspect, as shown in FIG.3B, projections 72 in the form of thermoplastic needles can be activatedto engage (e.g., grab onto) the tissue with an expanded tip anchor 75.The upper surface 30 of the tissue 25 and the undersurface 40 of thetissue can have the same characteristics depicted and described withrespect to FIGS. 2A-2B.

The use of micro or nanopillar patterning massively enhances theadhesive force as has been shown for Gecko feet dry adhesion models(Menguc et al Advanced Functional Materials 2012, 22, 1246-1254) whileat the same time improving the resolution to achieve a capture areadefined by the narrowest micropillar diameter. The laser energy canactivate the micropillars to become adhesive by various means as shownin FIGS. 3A-3B. By impregnating the micropillar with a gradient of a UVor IR absorbing dye the tip can be made to swell and conform to thetissue surface irregularities (FIG. 3A) selectively at the tip. Thediameter of the pillar strictly confines the region of resolution.Alternatively, the micropillar can be a micro needle with aconcentration of the dye in the tip that penetrates into the tissue andis then permanently anchored into the tissue (below the surface) by thelaser energy (FIG. 3B).

FIGS. 4A-4C show configurable aspects of hydrogel particles (e.g.,nanoparticles), which can be provided on at least one surface of theprojections 72. The hydrogel particles can be deformable and can beconfigured to modify the adhesive properties of the system. FIG. 4Ashows non-adhesive particles 90 that are shed from an activatedprojection 72 or tip of the micropatterned film 70, as the projectionexpands and contacts the tissue upper surface 30 but not the lowertissue surface 40. FIG. 4B shows schematically that the an adhesiveparticle can be made non-adhesive by breaking the shell 92 or polymermesh of the particle by dissociating crosslinks in the shell or polymermesh by interacting with an IR or UV laser beam. FIG. 4C shows analternative configuration for the particle wherein the shell has ahydrophobic non-adhesive character, and upon interaction with an IR orUV laser beam, the non-adhesive hydrophobic shell is shed to expose aninner adhesive core 94.

In various aspects, both the upper and lower surface of the tissue canbe in contact with surface with tunable adhesive properties. In oneaspect, the tip of the projection (e.g., micropillar) structure 72 canbe coated with nanoparticles or microparticles as shown in FIG. 4A. In afurther aspect, the slide surface can be coated with nanoparticles ormicroparticles as shown in FIGS. 2A-2C. The nanoparticles ormicroparticles provide a means of modifying the adhesive properties, andas shown, can be localized at one or both surfaces of the tissue sample25. For example, as shown in FIG. 4A, the tip of the projection (e.g.,micropillar) structure can be activated by the laser energy to shed anon-adhesive microparticle coating or to activate a microparticlecoating to become adhesive. In an aspect, an example of modifiablenanoparticles is hydrogel particles with reversible crosslinks that canopen up in response to activation (see FIG. 4C). Once open they canexpose an inner core 94 containing a covalently bound dye that isadhesive. Alternatively, in a further aspect, an adhesive particle withdissociating cross-links is converted to a non-adhesive material bybreaking of the shell 92 or polymer mesh using laser-activateddissociation as shown in FIG. 4B. In various aspects, nanoparticles ormicroparticles can also be coated onto the glass slide, thus provide atunable adhesive surface on the lower surface 40 of the tissue 25.

A. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of thedisclosure and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

An example of the micropatterned thermoplastic film of the presentdisclosure was prepared and characteristics are shown in FIGS. 5A-5F. Inthis example, the fabricated micropillars were formed on the surface ofthe film and impregnated with IR absorbing dyes. The tips of themicropillars can be made to be wetted (FIG. 5D) and the charge on thesurface of the tips and the base of the micropillars can bedifferentially charged by plasmon surface coating. The overall goal isto create a micro- or nano-structured modification of the standarddisposable components of conventional LCM systems, such as the ThermoArcturus LCM system. Although these materials can have other uses, aparticular use is to provide a significant improvement on performancethat can be sold as new disposables for the LCM instruments that alreadyexist throughout the world.

The LCM caps with pillar structures were fabricated utilizing a simplereplica molding procedure. Initially, utilizing standard softlithography methods, a negative block of polymer polydimethylsiloxane(PDMS) was created containing 10 μm deep wells of desired dimensions.Subsequently, the PDMS block was placed on a hot plate preheated to 125°C. and a blank LCM cap is placed in close contact with the patternedsurface of PDMS. A dead weight was placed on top of the cap to keep itpressed against the PDMS wells. After 15-20 minutes of replica molding,the composite device was allowed to cool down to room temperature andthe LCM cap containing pillar structures was separated from the PDMSblock.

A photomicrograph of a fabricated micropattern surface is shown in FIGS.5A and 5B. The photomicrograph was produced by an invertedphotolithography mold applied to the thermopolymer surface mounted on astandard cap (FIG. 5C) for LCM on a Thermo Arcturus Instrument. FIGS. 5Aand 5B show the uniformity of example micropillars that can befabricated in any desired density and size. FIG. 5D shows how the tipsof the micropillars can be made wetted by a minute drop of water whilethe base remains repulsive and non-wetted.

FIGS. 5E and 5F are representative microscopic images (100×magnification) of captured cells using the micropatterned surfaces ofFIGS. 5A and 5B. Cancer cells were collected from colorectal cancerinvading dense collagenous stroma within an 8 micron Hematoxylin stainedtissue section mounted on an uncharged microscope slide. Without the useof the micropatterned surface, the capture of tumor cells from this casewas zero percent. Both large and smaller micropatterns (5 microns (FIG.5E) versus 10 microns (FIG. 5F)) captured greater than 95% of thedesired tumor cells demonstrating the large improvement in captureefficiency afforded by the disclosed apparatus and method. Thus, incontrast to existing microdissection methods, it is contemplated thatthe micropatterned thermoplastic films disclosed herein can effectivelycapture human tumor cells within dense fibrotic collagenous stroma.

A similar strategy can be utilized to create needle shaped structuresinstead of cylindrical pillars. Alternatively, IR sensitive polymer canbe selectively deposited at the tip of PDMS needles. By fabricatingmolds containing conical wells, PDMS or plastic blocks with needleshaped structures can be easily created. Subsequently placing theseneedle shaped features in contact with a film of IR polymer at atemperature slightly higher than its liquid transition temperature willdeposit a layer of the IR material on PDMS needles.

Nanoparticles (and other particles) can be immobilized on projection(e.g., pillar) tips or other surface modification can be performed.Proteins of interest can be adsorbed onto the PDMS surface bymicrocontact printing for which complementary ligands can befunctionalized on nanoparticles. Placing a drop of nanoparticles on thefunctionalized surface can allow them to adhere specifically at the tipof projections (e.g., pillars or needles) where proteins werepredeposited.

Exemplary Aspects

In view of the described devices, systems, and methods and variationsthereof, herein below are described certain more particularly describedaspects of the invention. These particularly recited aspects should nothowever be interpreted to have any limiting effect on any differentclaims containing different or more general teachings described herein,or that the “particular” aspects are somehow limited in some way otherthan the inherent meanings of the language literally used therein.

Aspect 1: An apparatus comprising: a patterned thermoplastic filmhaving: a first surface; and a plurality of projections attached to andextending outwardly from the first surface, wherein the projections forma pattern on the thermoplastic film.

Aspect 2: The apparatus of aspect 1, wherein the projections extendoutwardly from the first surface of the patterned thermoplastic filmrelative to an axis that is substantially perpendicular to the firstsurface.

Aspect 3: The apparatus of aspect 1 or aspect 2, wherein the projectionsare continuous with, and integrally formed with, the first surface ofthe patterned thermoplastic film.

Aspect 4: The apparatus of any one of aspects 1-3, wherein the patternis an array of projections.

Aspect 5: The apparatus of any one of aspects 1-4, wherein at least oneof the projections has a consistent diameter.

Aspect 6: The apparatus of any one of aspects 1-5, wherein at least oneof the projections has a variable diameter.

Aspect 7: The apparatus of aspect 6, wherein at least one of theprojections is a microneedle that has a diameter that decreases movingaway from the first surface of the patterned thermoplastic film.

Aspect 8: The apparatus of aspect 6, wherein at least one of theprojections is a microsphere.

Aspect 9: The apparatus of aspect 8, wherein the microsphere comprises ahydrogel.

Aspect 10: The apparatus of any one of aspects 1-9, wherein eachprojection defines at least one tip, and wherein at least one tip of atleast one of the projections is coated with one or more particles.

Aspect 11: The apparatus of aspect 10, wherein the particles arenanoparticles.

Aspect 12: The apparatus of aspect 10, wherein the particles aremicroparticles.

Aspect 13: The apparatus of any one of aspects 10-12, wherein at leastone particle comprises a hydrogel.

Aspect 14: The apparatus of any one of aspects 10-13, wherein at leastone particle comprises dissociating cross-links.

Aspect 15: The apparatus of aspect 14, wherein the dissociatingcross-links dissociate upon irradiation with electromagnetic radiationin the UV or IR spectrum.

Aspect 16: The apparatus of any one of aspects 10-13, wherein at leastone particle comprises reversible cross-links.

Aspect 17: The apparatus of aspect 16, wherein the reversiblecross-links are cleaved upon irradiation with electromagnetic radiationin the UV or IR spectrum.

Aspect 18: The apparatus of any one of aspects 10-17, wherein at leastone particle comprises an outer shell and an inner core.

Aspect 19: The apparatus of aspect 18, wherein the inner core isadhesive with respect to a cell surface.

Aspect 20: The apparatus of aspect 18 or aspect 19, wherein the shell isnon-adhesive with respect to a cell surface.

Aspect 21: The apparatus of any one of aspects 10-17, wherein, in theabsence of irradiation with electromagnetic radiation in the UV or IRspectrum, at least one particle is adhesive with respect to a cellsurface.

Aspect 22: The apparatus of aspect 21, wherein the at least one particleis non-adhesive with respect to a cell surface upon irradiation withelectromagnetic radiation in the UV or IR spectrum.

Aspect 23: The apparatus of any one of aspects 1-22, wherein thepatterned thermoplastic film further comprises an adsorptive substance.

Aspect 24: The apparatus of aspect 23, wherein the adsorptive substanceis an infrared napthalocyanine dye.

Aspect 25: The apparatus of aspect 23, wherein the adsorptive substanceis tin(IV) 2,3-naphthalocyanine dichloride; silicon(IV)2,3-naphthalocyanine dihydroxide; silicon (IV) 2,3-naphthalocyaninedioctyloxide; or vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, or combinationsthereof.

Aspect 26: The apparatus of any one of aspects 1-25, wherein thepatterned thermoplastic film comprises an ethyl vinyl acetate polymer, avipolyurethane polymer; a polyvinyl acetate polymer; an ethylene-methylacrylate polymer; a polycarbonate polymer; an ethylene-vinyl alcoholcopolymer; a polypropylene polymer; or an expandable or general purposepolystyrene polymer, or combinations thereof.

Aspect 27: A method for laser capture microdissection, comprising thesteps of: (a) placing the apparatus of any one of aspects 1-26 incontact with a tissue sample, and (b) irradiating the apparatus incontact with the tissue sample with electromagnetic energy orwavelengths in the UV or IR spectrum.

Aspect 28: The method of aspect 27, wherein the patterned thermoplasticfilm contacts a first surface of the tissue sample, and wherein anopposed second surface of the tissue sample rests on a coating ofmicroparticles or nanoparticles that is positioned on an upper surfaceof a glass slide.

Aspect 29: A kit comprising the apparatus of any one of aspects 1-26,and instructions for the use of the apparatus in laser capturemicrodissection.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present disclosurewithout departing from the scope or spirit of the disclosure. Otherembodiments of the disclosure will be apparent to those skilled in theart from consideration of the specification and practice of thedisclosure disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the disclosure being indicated by the following claims.

What is claimed is:
 1. A method for laser capture microdissection,comprising the steps of: (a) placing a patterned thermoplastic film incontact with a tissue sample, the patterned thermoplastic film having afirst surface and a plurality of projections attached to and extendingoutwardly from the first surface, wherein the projections form a patternon the thermoplastic film; and (b) irradiating the patternedthermoplastic film in contact with the tissue sample withelectromagnetic energy or wavelengths in the UV or IR spectrum.
 2. Themethod of claim 1, wherein the patterned thermoplastic film contacts afirst surface of the tissue sample, and wherein an opposed secondsurface of the tissue sample rests on a coating of microparticles ornanoparticles that is positioned on an upper surface of a glass slide.3. The method of claim 1, wherein the projections extend outwardly fromthe first surface of the patterned thermoplastic film relative to anaxis that is substantially perpendicular to the first surface.
 4. Themethod of claim 1, wherein the projections are continuous with, andintegrally formed with, the first surface of the patterned thermoplasticfilm.
 5. The method of claim 1, wherein the pattern is an array ofprojections.
 6. The method of claim 1, wherein at least one of theprojections has a consistent diameter.
 7. The method of claim 1, whereinat least one of the projections has a variable diameter.
 8. The methodof claim 7, wherein at least one of the projections is a microneedlethat has a diameter that decreases moving away from the first surface ofthe patterned thermoplastic film.
 9. The method of claim 7, wherein atleast one of the projections is a microsphere.
 10. The method of claim9, wherein the microsphere comprises a hydrogel.
 11. The method of claim1, wherein each projection defines at least one tip, and wherein atleast one tip of at least one of the projections is coated with one ormore particles, wherein the particles are microparticles, nanoparticles,or combinations thereof.
 12. The method of claim 11, wherein at leastone particle comprises a hydrogel.
 13. The method of claim 11, whereinat least one particle comprises dissociating cross-links, wherein thedissociating cross-links dissociate upon irradiation withelectromagnetic radiation in the UV or IR spectrum.
 14. The method ofclaim 11, wherein at least one particle comprises reversiblecross-links, wherein the reversible cross-links are cleaved uponirradiation with electromagnetic radiation in the UV or IR spectrum. 15.The method of claim 11, wherein at least one particle comprises an outershell and an inner core.
 16. The method of claim 15, wherein the innercore is adhesive with respect to a cell surface, and wherein the shellis non-adhesive with respect to a cell surface.
 17. The method of claim11, wherein, at least one particle is: (a) adhesive with respect to acell surface in the absence of irradiation with electromagneticradiation in the UV or IR spectrum; and (b) non-adhesive with respect toa cell surface upon irradiation with electromagnetic radiation in the UVor IR spectrum.
 18. The method of claim 1, wherein the patternedthermoplastic film further comprises an adsorptive substance, andwherein the adsorptive substance is an infrared napthalocyanine dye;tin(IV) 2,3-naphthalocyanine dichloride; silicon(IV)2,3-naphthalocyanine dihydroxide; silicon (IV) 2,3-naphthalocyaninedioctyloxide; or vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, or combinationsthereof.
 19. The method of claim 1, wherein the patterned thermoplasticfilm comprises an ethyl vinyl acetate polymer, a vipolyurethane polymer;a polyvinyl acetate polymer; an ethylene-methyl acrylate polymer; apolycarbonate polymer; an ethylene-vinyl alcohol copolymer; apolypropylene polymer; or an expandable or general purpose polystyrenepolymer, or combinations thereof.